1. INTRODUCTION TO MICROBIOLOGY

2. MICROBIOLOGY
The study of organisms too small to be seen without magnification
Bacteria
Viruses
Fungi
Protozoa
Helminths (worms)
Algae
Some multicellular parasites

3. Pioneers of Microbiology
Robert Hooke, UK (1665)
Proposed the Cell Theory
Observed cork with crude microscope
All living things are composed of cells
Spontaneous generation
Some forms of life could arise spontaneously from non-living matter
Francesco Redi, IT (1668)
Redi’s experiments first to dispprove S.G.

4. Pioneers of Microbiology
Antoni van Leeuwenhoek, DE (1673)
First observed live microorganisms (animalcules)
Schleiden and Schwann, DE
Formulated Cell Theory: cells are the fundamental units of life and carry out all the basic functions of living things
Pasteur, FR and Tyndall, UK (1861)
Finally disproved S.G.

5. Pioneers of Microbiology
Louis Pasteur ( ), Chemist
Fermentation (1857)
Pasteurization: heat liquid enough to kill spoilage bacteria (1864)
Vaccine development – rabies
Proposed the germ theory of disease
Proposed aseptic techniques (prevent contamination by unwanted microbes)
Director of Pasteur Institute, Paris (1894)

6. Pioneers of Microbiology
Joseph Lister, UK (1867)
Used phenol (carbolic acid) to disinfect wounds
First aseptic technique in surgery
Robert Koch, DE (1876)
Postulates – Germ theory (1876)
Identified microbes that caused anthrax (1876), tuberculosis (1882) and cholera (1883)
Developed microbiological media & streak plates for pure culture (1881)

7. Koch’s Postulates
The specific causative agent must be found in every case of the disease.
The disease organism must be isolated from the lesions of the infected case and maintained in pure culture.
The pure culture, inoculated into a susceptible or experimental animal, should produce the symptoms of the disease.
The same bacterium should be re-isolated in pure culture from the intentionally infected animal.

8. Branches of Microbiology
Bacteriology: study of bacteria
Mycology: study of fungi
Parasitology: study of protozoa and parasitic worms
Virology: study of viruses
Beijerinck, NE: discovered intracellular reproduction of TMV; coined the term “virus” (1899)

9. Branches of Microbiology
Chemotherapy
Treatment of disease by using chemical means
Antibiotics produced naturally
Synthetic drugs
Paul Ehrlich (1878) – used arsenic compounds to fight disease – ‘magic bullet’
Immunology: study of immunity
Edward Jenner, UK: developed vaccination (1798)
Metchnikoff, RU: discovered phagocytes (1884)
Paul Ehrlich, DE: theory of immunity (1890)

10. Branches of Microbiology
Chemotherapy
Alexander Fleming, Scotland (1928) discovered penicillin
Selman Waksman, Ukraine (1944) discovered streptomycin
Problems
Toxicity of drugs => Selective toxicity
Resistance of bacteria to drugs

11. Branches of Microbiology
Recombinant DNA Technology
Recombinant DNA
Genetic engineering/biotechnology
Microbial genetics – mechanism by which microbes inherit genes
Molecular biology – structure and function (expression) of genes
Molecular epidemiology/diagnostics
Beadle and Tatum (1941) – demonstrated the relationship between genes and enzymes
Avery, MacLeod and McCarty (1944) – established that DNA (not protein) was the hereditary material
Lederberg and Tatum (1946) – discovered the transmissibility of the genetic material between bacteria via conjugation.
Watson and Crick (1953) – proposed the model for the structure and replication of DNA
Jacob and Monod (1961) – discovered messenger RNA

12. MICROBES ARE INVOLVED IN
Nutrient production & energy flow
Decomposition (bioremediation)
Production of foods
Production of drugs & vaccines
Genetic engineering
Causing disease

13. MICROORGANISM CLASSIFICATION
Microorganisms and all other living organisms are classified as prokaryotes or eukaryotes.
Prokaryotes are probably the smallest living organisms, ranging in size from 0.15 um (mycoplasmas) to about 2.0 um (many of the bacteria).
Viruses and subparticles such as prions are considered neither prokaryotes nor eukaryotes because they lack the characteristics of living things, except the ability to replicate.

14. Prokaryotes Cell Wall Teichoic Acids LPS Endospores Circular DNA
Plasmids

16. Classification Schemes
Traditionally these have been inferred on the basis of morphologic or biochemical characteristics.
Schemes have recently been revised based on the degree of genetic (DNA, RNA) similarity between different species.
Genus and species are of primary importance in designating a microorganism.
The correct format for naming an organism is genus (capitalized, italicized, or underlined), species (lowercase, italicized, or underlined): Escherichia coli (abbreviation, E. coli).

17. Size of Bacteria Average bacterial cell diameter is 0.5 - 2.0 um.
Surface Area ~12 square um
Volume is ~4 cubic um
Surface Area to Volume is 3:1
Typical Eukaryote Cell SA/ Vol. is 0.3:1
Food enters through SA, quickly reaches all parts of bacteria
Eukaryotes need structures & organelles

18. Shapes of Bacteria Spherical (Cocci) Rod Shape (Bacilli) Coccobacilli
Chain = Streptococcus
Cluster = Staphylococcus
Rod Shape (Bacilli)
Chain = Streptobacillus
Coccobacilli
Comma shape (Vibrios)
Spirillum
Spirochete
Square
Star

23. Bacterial Structures Flagella Pili Capsule Cell Wall
- Lipopolysaccharides
- Teichoic Acids
Plasma Membrane
Cytoplasm
- Genetic materials
- Ribosomes
Inclusions
Spores

24. Extracellular Polymeric Substance (EPS)
Polysaccharide on external surface: Capsule, Glycocalyx, or Slime (Antigen)
EPS does not take ordinary stains, is not necessary for survival of the cell, and may be lost upon continuous cultivation
Adherence of bacteria to surfaces (S. mutans and enamel of teeth)
Prevention of Phagocytosis (Complement cannot penetrate sugars)

25. The Cell Wall
The cell wall of bacteria is a complex, semi-rigid structure that is made up of peptidoglycan (mucopeptide or murein), responsible for the shape of the cell.
It differs between gram positive and gram negative bacteria
In most gram-positive bacteria the cell wall consists of many layers of peptidoglycan forming a thick rigid structure.
By contrast, gram-negative cell walls contain only one (or very few) layers of peptidoglycan.

28. Cell Wall Peptidoglycan Polymer (amino acids + sugars)
Unique to bacteria
Sugars
- N- acetylglucosamine (NAG)
- N- acetylmuramic acid (NAM)
D form of Amino acids used not L form
Hard to break down D form
Amino acids cross link NAG & NAM

31. Cell Wall Gram positive Bacteria - Peptidoglycan
- Teichoic ( ribitol or glycerol residues) and Teichuronic (Sugar acids) acids; wall and membrane Teichoic acids supply cell with magnesium.
- Polysaccharide
Gram negative Bacteria
- Lipoprotein
- Outer membrane
- Periplasmic space

32. The Outer Membrane
Gram-negative cells possess an outer membrane that is composed of lipoproteins, lipopolysaccharides, and phospholipids.
The outer membrane helps some organisms evade phagocytosis, provides a barrier to certain antibiotics, and confers properties of virulence (endotoxin).

34. Lipopolysaccharide (LPS)
Endotoxin or Pyrogen
Fever causing
Toxin nomenclature
Endo - part of bacteria
Exo - excreted into environment
Structure
Lipid A
Polysaccharide
O Antigen
Gram negative bacteria only
Removed by Alcohol/Acetone

36. LPS (cont’d.) Appearance of Colonies
Mucoid = Smooth (lots of LPS or capsule)
Dry = Rough (little LPS or capsule)
O Antigen of Salmonella and E. coli
2,000 different O Ags of Salmonella
100’s different O Ags of E. coli
E. coli O157
O Ags differ in Sugars, not Lipid A

37. LPS (cont’d) Functions Heat Resistant; hard to remove
Toxic; kills mice, pigs, humans
G - ve septicemia; death due to LPS
Pyrogen; causes fever
DPT vaccination always causes fevers
Adjuvant; stimulates immunity
Heat Resistant; hard to remove
Detection (all topical & IV products)
Rabbits (measure fever)
Horse shoe crab (Amoebocytes Lyse in presence of LPS)

38. Cell Wall Summary Unique to bacteria 20-40% of bacterial cell weight
Determines shape of bacteria
Prevents osmotic rupture
Target for some antibiotics (Penicillin)

39. Cell Membrane
The plasma membrane encloses the cytoplasm of the cell and provides selective permeability for nutrients to enter.
Phospholipid Bilayer
Water can penetrate
Flexible
Not strong, ruptures easily
Osmotic Pressure created by cytoplasm

42. Cytoplasmic Structures
80% Water {20% Salts-Proteins)
DNA is a single long circular molecule of double-stranded DNA “bacterial chromosome”.
More efficient; grows quicker
Mutations allow adaptation to environment quicker
Plasmids; small circular transferable, double-stranded DNA molecules
Antibiotic Resistance
Bacteria also contain transposons
Ribosomes function as the site of protein synthesis.
No organelles (Mitochondria, Golgi, etc.)

43. Appendages of Bacteria
Some bacteria have flagella which are long filamentous appendages that can propel the cell.
Many gram-negative bacteria possess hair-like appendages that are used for attachment rather than for motility. These are divided into two types, fimbriae and pili.
Fimbriae enable a bacterial cell to adhere to surfaces (including other cells) while pili join bacterial cells in preparation for the transfer of DNA from one cell to another.

44. Flagella Motility - movement Swarming occurs with some bacteria
Spread across Petri Dish
Proteus species most evident
Arrangement basis for classification
Monotrichous; 1 flagella
Lophotrichous; tuft at one end
Amphitrichous; both ends
Peritrichous; all around bacteria

46. Mono- or Lophotrichorus

49. Pilli Short protein appendages Adherence of bacteria to surfaces
smaller than flagella
Adherence of bacteria to surfaces
E. coli has numerous types
K88, K99, F41, etc.
Antibodies to will block adherence
F- Pilus; used in conjugation
Exchange of genetic information

50. F- Pilus for Conjugation

51. Endospores
When essential nutrients are depleted, certain gram positive bacteria (e.g., Clostridium and Bacillus), form “resting” cells called endospores.
These endospores contain condensed nuclear material and protein and can survive extreme heat, lack of water, and exposure to toxic chemicals.
When growth conditions permit, the cell will germinate into a dividing bacterium.

52. Endospores Resistant structure Takes time and energy to make spores
Heat, irradiation, cold
Boiling >1 hr
Takes time and energy to make spores
Location important in classification
Central, Subterminal, Terminal
Bacillus stearothermophilus -spores
Used for quality control of heat sterilization equipment
Bacillus anthracis - spores
Used in biological warfare